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An aerobic endospore-forming bacillus (NVH 391-98(T)) was isolated during a severe food poisoning outbreak in France in 1998, and four other similar strains have since been isolated, also mostly from food poisoning cases. Based on 16S rRNA gene sequence similarity, these strains were shown to belong to the Bacillus cereus Group (over 97% similarity with the current Group species) and phylogenetic distance from other validly described species of the genus Bacillus was less than 95%. Based on 16S rRNA gene sequence similarity and MLST data, these novel strains were shown to form a robust and well-separated cluster in the B. cereus Group, and constituted the most distant cluster from species of this Group. Major fatty acids (iso-C(15:0), C(16:0), iso-C(17:0), anteiso-C(15 : 0), iso-C(16:0), iso-C(13:0)) supported the affiliation of these strains to the genus Bacillus, and more specifically to the B. cereus Group. NVH 391-98(T) taxon was more specifically characterized by an abundance of iso-C(15:0) and low amounts of iso-C(13:0) compared with other members of the B. cereus Group. Genome similarity together with DNA-DNA hybridization values and physiological and biochemical tests made it possible to genotypically and phenotypically differentiate NVH 391-98(T) taxon from the six current B. cereus Group species. NVH 391-98(T) therefore represents a novel species, for which the name Bacillus cytotoxicus sp. nov. is proposed, with the type strain NVH 391-98(T) (= DSM 22905(T) = CIP 110041(T)).

Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases and results from the ingestion of staphylococcal enterotoxins (SEs) preformed in food by enterotoxigenic strains of Staphylococcus aureus. To date, more than 20 SEs have been described: SEA to SElV. All of them have superantigenic activity whereas half of them have been proved to be emetic, representing a potential hazard for consumers. This review, divided into four parts, will focus on the following: (1) the worldwide story of SFP outbreaks, (2) the characteristics and behaviour of S. aureus in food environment, (3) the toxinogenic conditions and characteristics of SEs, and (4) SFP outbreaks including symptomatology, occurrence in the European Union and currently available methods used to characterize staphylococcal outbreaks., (© European Union 2011 Published by Blackwell Publishing Ltd.)

At the end of 2009, six food poisoning outbreaks caused by staphylococci were reported in France. Soft cheese made from unpasteurized milk was found to be the common source of the outbreaks. Staphylococcal enterotoxin type E was identified and quantified in the cheese using both official and confirmatory methods of the European Union Reference Laboratory (EU-RL). To our knowledge, this is the first report of food poisoning outbreaks caused by staphylococcal enterotoxin type E in France.

Bacillus cereus is found in food, soil, and plants, and the ability to cause food-borne diseases and opportunistic infection presumably varies among strains. Therefore, measuring harmful toxin production, in addition to the detection of the bacterium itself, may be key for food and hospital safety purposes. All previous studies have focused on the main known virulence factors, cereulide, Hbl, Nhe, and CytK. We examined whether other virulence factors may be specific to pathogenic strains. InhA1, NprA, and HlyII have been described as possibly contributing to B. cereus pathogenicity. We report the prevalence and expression profiles of these three new virulence factor genes among 57 B. cereus strains isolated from various sources, including isolates associated with gastrointestinal and nongastrointestinal diseases. Using PCR, quantitative reverse transcriptase PCR, and virulence in vivo assays, we unraveled these factors as potential markers to differentiate pathogenic from nonpathogenic strains. We show that the hlyII gene is carried only by strains with a pathogenic potential and that the expression levels of inhA1 and nprA are higher in the pathogenic than in the nonpathogenic group of strains studied. These data deliver useful information about the pathogenicity of various B. cereus strains.

Staphylococcal poisoning is a common food-borne disease for which immunoassays to detect enterotoxins were developed, but these assays often lead to false diagnoses due to interferences or lack of specificity. Absolute quantitative mass spectrometry was for the first time successfully applied to an investigation of a staphylococcal outbreak due to coconut pearls.

Aim: Immunological tools used to detect staphylococcal enterotoxins (SEs) in foods are numerous. The aim of this study was to evaluate, on naturally contaminated milk product samples, the performance of the Vidas SET2, in comparison to the Transia plate SET., Methods and Results: The Vidas SET2 was compared with the Transia plate SET on supernatants of Staphylococcus aureus isolates and on naturally contaminated milk products. It is noteworthy that when using IgG rabbit treatment, both kits can be considered as equivalent to detect enterotoxins in naturally contaminated milk products., Conclusions: This study demonstrated that the Vidas SET2 performance is similar to that of Transia plate SET kit, when a rabbit IgG treatment step is used before detection step. This additional treatment significantly decreased, from 42% to 8%, the rate of positive deviations observed using the Transia plate SET detection kit., Significance and Impact of the Study: The Vidas SET2 was clearly found as more specific, when no preliminary rabbit IgG treatment was used, and which results in a better workflow when a large number of samples have to be analysed within a few days. Considering the results obtained, the Vidas SET2 detection kit can be used to assess the safety of milk products for SEs.

Enterotoxins produced by Staphylococcus aureus are responsible for staphylococcal food-poisoning outbreaks (SFPO). In France, SFPO are the second cause of food-borne diseases after Salmonella. However, very little is known about the strains involved. The objective of this study was to characterize the staphylococcal strains related to these SFPO through phenotypic and genotypic analyses. A total of 178 coagulase-positive staphylococcal isolates recovered from 31 SFPO (1981-2002) were screened through biotyping. Thirty-three strains representative of the different biotypes in each SFPO were further examined for SmaI macrorestriction-type, phage-type, resistance to various antimicrobial drugs, presence of staphylococcal enterotoxin (se) genes sea to sei, and production of enterotoxins SEA to SED. All these 33 strains were identified as S. aureus species: 27 were of human biotypes and six ovine or non-host-specific biotypes. Most (74.1%) strains reacted with group III phages. Eleven strains were resistant to at least two classes of antibiotics and among them, two were resistant to methicillin. Twenty-nine strains carried one or several of the eight se genes tested; the gene sea was most common (n=23), and often linked to sed (n=12) or seh (n=5). The novel se genes seg-i were in all cases associated with se genes sea to sed except for one strain which carried only seg and sei. Pulsed-Field Gel Electrophoresis (PFGE) of SmaI macrorestriction digests of the 33 strains discriminated 32 PFGE patterns grouped into nine biotype-specific clusters. All five strains carrying sea and seh were grouped together into the same sub-cluster. Three of the four se-gene-negative strains were in one PFGE cluster: all four should be tested for se genes not included in this study and, if negative, be further investigated for the presence of unidentified SEs.

The methods of European and International Organisations for Standardization for the enumeration of coagulase-positive staphylococci (CPS, Staphylococcus aureus and other species) described in EN ISO 6888 Part 1 and Part 2: 1999 were validated by order of the European Commission (Standards, Measurement and Testing Fourth Framework Programme Project SMT4-CT96-2098). EN ISO 6888-1 prescribes the use of Baird-Parker (BP) agar whereas EN ISO 6888-2 prescribes the use of Rabbit Plasma Fibrinogen Agar (RPFA). The objective was to determine the precision of each method in terms of repeatability (r) and reproducibility (R) using three different food types inoculated with various levels of S. aureus and a typical background flora. The results are intended for publication in the associated standards. Cheese, meat and dried egg powder were examined by 24 laboratories from 16 countries in Europe. Each participant received eight test materials per food type: blind duplicates at four inoculum levels (0, 10(3), 10(4) to 10(5), 10(5) to 10(6) cfu/g). In addition, two reference materials (RM) (capsules containing milk powder inoculated with S. aureus) were included in the study. All test materials were subjected to stringent homogeneity and stability testing before being used in the collaborative trial. Two statistical methods were used to calculate the precision parameters. Draft EN ISO 16140: 2000 method appeared more appropriate to the case of microbiological data than ISO 5725-2: 1994 method and was retained to calculate the precision data. Concerning EN ISO 6888-1, overall values for repeatability (r) when used with food test materials was r=log(10) 0.28 (expressed as an absolute difference between log(10)-transformed test results). For the reference materials, r=log(10) 0.19. Overall values for reproducibility (R) when used with food test materials were R=log(10) 0.43. For the reference materials, R=log(10) 0.39. Concerning EN ISO 6888-2, overall values for repeatability (r) when used with food test materials were r=log(10) 0.22. For the reference materials, r=log(10) 0.17. Overall values for reproducibility (R) when used with food test materials were R=log(10) 0.33. For the reference materials, R=log(10) 0.31. These results were presented to the ISO technical committee and to the Comité Européen de Normalisation (CEN). Both committees agreed to incorporate the precision data obtained with food materials as two amendments to EN ISO 6888-1 and -2, and to give an equal status to each part of the standard.

Aims: To examine whether pulsed-field gel electrophoresis (PFGE) of DNA macro-restriction fragments could provide better discrimination among the different biotypes previously described within the species Staphylococcus aureus than the traditional biochemical approach., Methods and Results: Seventy three Staph. aureus strains from various sources (human, animal or food origin) and belonging to eight biotypes, including the poultry-like biotype, tentatively designated as an 'abattoir' biotype, were genotyped by PFGE after SmaI digestion of DNA. The PFGE patterns were compared using the average linkage matching method (UPGMA) with the Dice coefficient. A total of 61 PFGE patterns were observed, showing between 31 and 100% similarity. In most cases, strains with the same biotype were grouped specifically into one, two or three separate sub-clusters. Strains from the 'abattoir' biotype were clustered in one separate sub-cluster., Conclusions: The PFGE typing is useful to distinguish the traditional biotypes of Staph. aureus and has a more discriminatory power than the biochemical typing., Significance and Impact of the Study: The PFGE typing confirms the 'abattoir' biotype as a separate group on a genetic level and is well suited to investigate modes of staphylococcal contamination of food.

A study was carried out to estimate the proportion of diseases due to milk and milk products among food-borne diseases recorded in France and in other countries since 1980. Particular attention was given to whether the milk involved was heat-treated or not. Four etiologic agents were considered: Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, and pathogenic Escherichia coli. An overview of food-borne disease annual reports from seven countries indicated that milk and milk products were implicated in 1-5% of the total bacterial outbreaks; however, details about the type of product and milk involved were usually not provided. When considering 60 outbreaks and four single cases described in the literature and implicating milk and milk products, confirmed or suspected food vehicles were distributed as follows: milk, 39.1%, cheese, 53.1%, other milk products, 7.8%. Overall, 32.8% of the food vehicles were made from pasteurised milk; 37.5% from raw milk; 10.9% from milk stated as "unpasteurised"; and 18.8% from unspecified milk. Salmonella spp. were responsible for 29 outbreaks, L. monocytogenes for 10 outbreaks and four well-documented single cases, pathogenic E. coli for 11 outbreaks, and S. aureus for 10 outbreaks. Analysis of unpublished data about food-borne disease outbreaks, listeriosis excluded, collected by the coordinator of the French surveillance system from 1992 to 1997, revealed 69 documented outbreaks for which milk and milk products were confirmed as the vehicle by the isolation of the etiologic agent. The food vehicles were distributed as follows: milk, 10%; cheese, 87%; others, 3%. UHT milk accounted for 1.5%, raw milk and raw milk products for 48%, and milk and milk products from unspecified milk for 50.5% of the 69 outbreaks. S. aureus was by far the most frequent pathogen associated with these outbreaks (85.5% of the outbreaks), followed by Salmonella (10.1%). This study demonstrates the limitations of the surveillance systems and the difficulties in estimating the contribution of milk and milk products to food-borne diseases. In particular, it was not possible to find out in many outbreaks what heat treatment, if any, the milk had undergone.

A cytotoxin (CytK) has been isolated from a Bacillus cereus strain that caused a severe food poisoning outbreak killing three people. A protein of 34 kDa was highly cytotoxic, and the addition of other secreted proteins gave no synergistic effect. CytK was also necrotic and haemolytic. No known B. cereus enterotoxins were produced by this strain. A DNA sequence from 1.8 kb upstream to 0.2 kb downstream of the toxin gene was sequenced. The deduced amino acid sequence of the toxin showed similarity to Staphylococcus aureus leucocidins, gamma-haemolysin and alpha-haemolysin, Clostridium perfringens beta-toxin and B. cereus haemolysin II, all belonging to a family of beta-barrel channel-forming toxins. There was no sequence similarity between CytK and enterotoxins of B. cereus. The upstream sequence contained a partial sequence of a putative histidine kinase gene. A recognition site for PlcR, which regulates the transcription of enterotoxins HBL and Nhe of B. cereus, was found in the promoter region of the toxin. This new cytotoxin may be responsible for a disease that is similar to, although not as severe as, the necrotic enteritis caused by the beta-toxin of C. perfringens type C.

Milk and dairy products harbour a natural microbial flora and/or other micro-organisms, which vary within the wide range of products available on the French market. The origin of contamination by pathogenic bacteria varies with the type of product and the mode of production and processing. Contamination of milk and dairy products by pathogenic micro-organisms can be of endogenous origin, following excretion from the udder of an infected animal. Contamination may also be of exogenous origin, through direct contact with infected herds or through the environment (e.g. water, personnel). Treatment and processing of milk can inhibit or encourage the multiplication of micro-organisms. The authors describe the relevant aspects of bacterial physiology and ecology, the occurrence of bacteria in dairy products, and the public health significance for each of the principal micro-organisms found in such products. Bacteria most frequently involved are mycobacteria, Brucella sp., Listeria monocytogenes, Staphylococcus aureus and enterobacteria (including toxigenic Escherichia coli and Salmonella). At present, systems of testing and surveillance are required for the control of pathogenic bacteria in milk and dairy products, as specified by regulations currently being developed for all countries in the European Union. Preventive measures should take into account the well-established facts concerning the potential microbiological impact of pathogenic bacteria on milk and dairy products. There should be increased recourse to risk analysis methods to assess the threat to the consumer with regard to the presence of pathogenic bacteria in food.

To evaluate a 16S rRNA gene probe for the identification of staphylococcal species and subspecies, we have augmented previous studies involving 12 staphylococcal species by analysing the remaining 16 species currently classified in the genus Staphylococcus. HindIII- and EcoRI-restricted DNA of isolates from validly described species of Staphylococcus was probed with radiolabelled plasmid pBA2 containing 16S rDNA from Bacillus subtilis. The Dice coefficient was used to assess similarity between the 74 HindIII- and the 81 EcoRI-hybridization patterns obtained from a total of 271 isolates belonging to 31 staphylococcal taxa (28 species, of which three include two subspecies). The use of HindIII yielded a better discrimination of the staphylococci than the use of EcoRI. All of the isolates belonging to the same species or subspecies, except S. hyicus isolates, were recovered as homogeneous clusters using their HindIII hybridization patterns. The phenotypically close taxa were clearly distinguished. Thus, the method presented in this study constitutes a powerful tool for the identification of taxa within the genus Staphylococcus.

Dot blot hybridization was used to screen 820 staphylococci for the presence of the gene coding for TSST-1. The DNA of 33 strains among 70 Staph. aureus strains isolated from suspected toxic shock syndrome (TSS) cases hybridized with the probe. These results agreed perfectly with those obtained with a phenotypic method (immunodiffusion). Among 608 Staph. aureus strains isolated over a period of one month from hospitalized patients, 66 (11%) hybridized with the probe; of these strains, 64 (97%) were found to produce TSST-1 in vitro. None of 145 coagulase-negative staphylococcal strains harboured DNA hybridizing with the probe. The data indicate that this genotypic assay is suitable for epidemiological studies.

The rRNA gene restriction pattern sof 110 strains belonging to 12 staphylococcal species have been determined. The strains, isolated from various sources, were epidemiologically unrelated. Total DNA was cleaved with restriction enzymes HindIII and EcoRI, electrophoretically separated and probed with radiolabelled 16S rDNA from Bacillus subtilis inserted in a plasmid vector, pBR322. Fourty-four distinct HindIII patterns and 44 distinct EcoRI patterns were observed. Strains belonging to different species had different patterns. Although distinct patterns were also observed with some species, a core of common bands could be discerned within each species or subspecies. Analysis of the patterns revealed two taxa in Staphylococcus xylosus which were not evident using phenotypic characteristics. Of 18 strains which were difficult to identify using phenotypic schemes, 15 showed patterns typical of known species. The three remaining atypical strains showed unusual patterns and may belong either to a known species, not included in the study, or to a new species. Since various patterns were observed within some species (e.g.S.aureus and S. epidermidis), rRNA gene restriction patterns may have epidemiological, as well as taxonomic interest.